Delayed-release formulation for reducing the frequency of urination and method of use thereof

ABSTRACT

Methods and compositions for reducing the frequency of urination are disclosed. One method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an analgesic agent formulated in a delayed-release formulation. Another method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising multiple active ingredients. Yet another method comprises administering to a subject in need thereof an effective amount of a diuretic followed with another administration of a pharmaceutical composition comprising an analgesic agent.

This application is a continuation application of U.S. patentapplication Ser. No. 13/847,940, filed Mar. 20, 2013, which is acontinuation application of U.S. patent application Ser. No. 13/560,665,filed Jul. 27, 2012, now U.S. Pat. No. 8,445,011, which is acontinuation application of U.S. patent application Ser. No. 13/487,343,filed Jun. 4, 2012, which is a continuation-in-part application of U.S.application Ser. No. 13/423,949, filed on Mar. 19, 2012, now U.S. Pat.No. 8,236,856, which is a continuation-in-part of U.S. patentapplication Ser. No. 13/343,349, filed on Jan. 4, 2012, which is acontinuation-in-part application of U.S. patent application Ser. No.12/956,634, filed on Nov. 30, 2010, which claims priority to 61/362,374filed on Jul. 8, 2010. The entirety of the aforementioned applicationsis incorporated herein by reference.

FIELD

The present application generally relates to methods and compositionsfor inhibiting the contraction of muscles and, in particular, to methodsand compositions for inhibiting the contraction of smooth muscles of theurinary bladder.

BACKGROUND

The detrusor muscle is a layer of the urinary bladder wall made ofsmooth muscle fibers arranged in spiral, longitudinal, and circularbundles. When the bladder is stretched, this signals the parasympatheticnervous system to contract the detrusor muscle. This encourages thebladder to expel urine through the urethra.

For the urine to exit the bladder, both the autonomically controlledinternal sphincter and the voluntarily controlled external sphinctermust be opened. Problems with these muscles can lead to incontinence. Ifthe amount of urine reaches 100% of the urinary bladder's absolutecapacity, the voluntary sphincter becomes involuntary and the urine willbe ejected instantly.

The human adult urinary bladder usually holds about 300-350 ml of urine(the working volume), but a full adult bladder may hold up to about 1000ml (the absolute volume), varying among individuals. As urineaccumulates, the ridges produced by folding of the wall of the bladder(rugae) flatten and the wall of the bladder thins as it stretches,allowing the bladder to store larger amounts of urine without asignificant rise in internal pressure.

In most individuals, the desire to urinate usually starts when thevolume of urine in the bladder reaches around 200 ml. At this stage itis easy for the subject, if desired, to resist the urge to urinate. Asthe bladder continues to fill, the desire to urinate becomes strongerand harder to ignore. Eventually, the bladder will fill to the pointwhere the urge to urinate becomes overwhelming, and the subject will nolonger be able to ignore it. In some individuals, this desire to urinatestarts when the bladder is less than 100% full in relation to itsworking volume. Such increased desire to urinate may interfere withnormal activities, including the ability to sleep for sufficientuninterrupted periods of rest. In some cases, this increased desire tourinate may be associated with medical conditions such as benignprostate hyperplasia or prostate cancer in men, or pregnancy in women.However, increased desire to urinate also occurs in individuals, bothmale and female, who are not affected by another medical condition.

Accordingly, there exists a need for compositions and methods for thetreatment of male and female subjects who suffer from a desire tourinate when the bladder is less than 100% full of urine in relation toits working volume. Said compositions and methods are needed for theinhibition of muscle contraction in order to allow in said subjects thedesire to urinate to start when the volume of urine in the bladderexceeds around 100% of its working volume.

SUMMARY

One aspect of the present application relates to a method for reducingthe frequency of urination. In one embodiment, the method comprisesadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising a first analgesic agent selectedfrom the group consisting of aspirin, ibuprofen, naproxen sodium, andacetaminophen, wherein the pharmaceutical composition is formulated in adelayed-release formulation and wherein said first analgesic agent isadministered orally at a daily dose of 5 mg to 2000 mg.

In another embodiment, the method comprises administering to a subjectin need thereof an effective amount of a pharmaceutical compositioncomprising a plurality of active ingredients, wherein the plurality ofactive ingredients comprises (1) one or more analgesic agents and/or (2)one or more antimuscarinic agents and wherein the one or more analgesicagents are administered orally at a combined daily dose of 5 mg to 2000mg. In some embodiments, the one or more analgesic agents are selectedfrom the group consisting of aspirin, ibuprofen, naproxen sodium andacetaminophen. Examples of the antimuscarinic agents include, but arenot limited to, oxybutynin, solifenacin, darifenacin, fesoterodine,tolterodine, trospium and atropine.

Another aspect of the present application relates to a pharmaceuticalcomposition, comprising: one or more analgesic agents selected from thegroup consisting of aspirin, ibuprofen, naproxen, and acetaminophen; oneor more antidiuretics; and a pharmaceutically acceptable carrier,wherein at least one of said one or more analgesic agents is formulatedfor delayed-release.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are diagrams showing that analgesics regulate expressionof co-stimulatory molecules by Raw 264 macrophage cells in the absence(FIG. 1A) or presence (FIG. 1B) of LPS. Cells were cultures for 24 hrsin the presence of analgesic alone or together with Salmonellatyphyiurium LPS (0.05 μg/ml). Results are mean relative % of CD40+CD80+cells.

DETAILED DESCRIPTION

The following detailed description is presented to enable any personskilled in the art to make and use the invention. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present invention. However, it will be apparent toone skilled in the art that these specific details are not required topractice the invention. Descriptions of specific applications areprovided only as representative examples. The present invention is notintended to be limited to the embodiments shown, but is to be accordedthe broadest possible scope consistent with the principles and featuresdisclosed herein.

As used herein, the term “effective amount” means an amount necessary toachieve a selected result.

As used herein, the term “analgesic” refers to agents, compounds ordrugs used to relieve pain and inclusive of anti-inflammatory compounds.Exemplary analgesic and/or anti-inflammatory agents, compounds or drugsinclude, but are not limited to, the following substances: non-steroidalanti-inflammatory drugs (NSAIDs), salicylates, aspirin, salicylic acid,methyl salicylate, diflunisal, salsalate, olsalazine, sulfasalazine,para-aminophenol derivatives, acetanilide, acetaminophen, phenacetin,fenamates, mefenamic acid, meclofenamate, sodium meclofenamate,heteroaryl acetic acid derivatives, tolmetin, ketorolac, diclofenac,propionic acid derivatives, ibuprofen, naproxen sodium, naproxen,fenoprofen, ketoprofen, flurbiprofen, oxaprozin; enolic acids, oxicamderivatives, piroxicam, meloxicam, tenoxicam, ampiroxicam, droxicam,pivoxicam, pyrazolon derivatives, phenylbutazone, oxyphenbutazone,antipyrine, aminopyrine, dipyrone, coxibs, celecoxib, rofecoxib,nabumetone, apazone, indomethacin, sulindac, etodolac, isobutylphenylpropionic acid, lumiracoxib, etoricoxib, parecoxib, valdecoxib,tiracoxib, etodolac, darbufelone, dexketoprofen, aceclofenac,licofelone, bromfenac, pranoprofen, loxoprofen, piroxicam, nimesulide,cizolirine,3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one,meloxicam, lornoxicam, d-indobufen, mofezolac, amtolmetin, pranoprofen,tolfenamic acid, flurbiprofen, suprofen, oxaprozin, zaltoprofen,alminoprofen, tiaprofenic acid, pharmacological salts thereof, hydratesthereof, and solvates thereof.

As used herein, the terms “coxib” and “COX inhibitor” refer to acomposition of compounds that is capable of inhibiting the activity orexpression of COX2 enzymes or is capable of inhibiting or reducing theseverity, including pain and swelling, of a severe inflammatoryresponse.

The urinary bladder has two important functions: storage of urine andemptying. Storage of urine occurs at low pressure, which implies thatthe detrusor muscle relaxes during the filling phase. Emptying of thebladder requires a coordinated contraction of the detrusor muscle andrelaxation of the sphincter muscles of the urethra. Disturbances of thestorage function may result in lower urinary tract symptoms, such asurgency, frequency, and urge incontinence, the components of theoveractive bladder syndrome. The overactive bladder syndrome, which maybe due to involuntary contractions of the smooth muscle of the bladder(detrusor) during the storage phase, is a common and underreportedproblem, the prevalence of which has only recently been assessed.

One aspect of the present application relates to a method for reducingthe frequency of urination by administering to a person in need thereofa pharmaceutical composition formulated in a delayed-releaseformulation. The pharmaceutical composition comprises one or moreanalgesic agents and, optionally, one or more antimuscarinic agents.

As used herein, the term “delayed-release” refers to a medication thatdoes not immediately disintegrate and release the active ingredient(s)into the body. In some embodiments, the term “delayed-release” is usedwith reference to a drug formulation having a release profile in whichthere is a predetermined delay in the release of the drug followingadministration. In some embodiments, the delayed-release formulationincludes an enteric coating, which is a barrier applied to oralmedication that prevents release of medication before it reaches thesmall intestine. Delayed-release formulations, such as enteric coatings,prevent drugs having an irritant effect on the stomach, such as aspirin,from dissolving in the stomach. Such coatings are also used to protectacid-unstable drugs from the stomach's acidic exposure, delivering theminstead to a basic pH environment (intestine's pH 5.5 and above) wherethey do not degrade, and give their desired action.

The term “pulsatile release” is a type of delayed-release, which is usedherein with reference to a drug formulation that provides rapid andtransient release of the drug within a short time period immediatelyafter a predetermined lag period, thereby producing a “pulsed” plasmaprofile of the drug after drug administration. Formulations may bedesigned to provide a single pulsatile release or multiple pulsatilereleases at predetermined time intervals following administration.

Most enteric coatings work by presenting a surface that is stable at thehighly acidic pH found in the stomach, but breaks down rapidly at a lessacidic (relatively more basic) pH. Therefore, an enteric coated pillwill not dissolve in the acidic juices of the stomach (pH˜3), but theywill in the alkaline (pH 7-9) environment present in the smallintestine. Examples of enteric coating materials include, but are notlimited to, methyl acrylate-methacrylic acid copolymers, celluloseacetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetatesuccinate), polyvinyl acetate phthalate (PVAP), methylmethacrylate-methacrylic acid copolymers, sodium alginate and stearicacid.

In some embodiments, the pharmaceutical composition is orallyadministered from a variety of drug formulations designed to providedelayed-release. Delayed oral dosage forms include, for example,tablets, capsules, caplets, and may also comprise a plurality ofgranules, beads, powders or pellets that may or may not be encapsulated.Tablets and capsules represent the most convenient oral dosage forms, inwhich case solid pharmaceutical carriers are employed.

In a delayed-release formulation, one or more barrier coatings may beapplied to pellets, tablets, or capsules to facilitate slow dissolutionand concomitant release of drugs into the intestine. Typically, thebarrier coating contains one or more polymers encasing, surrounding, orforming a layer, or membrane around the therapeutic composition oractive core.

In some embodiments, the active agents are delivered in a formulation toprovide delayed-release at a pre-determined time followingadministration. The delay may be up to about 10 minutes, about 20minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, or longer.

A delayed-release composition may comprise 100% of the total dosage of agiven active agent administered in a single unit dose. Alternatively, adelayed-release composition may be included as a component in a combinedrelease profile formulation may provide about 30-95% of the total dosageof the active agent(s) to be delivered by the pharmaceuticalformulation. For example, the immediate-release component may provideabout 5-70%, or about 50% of the total dosage of the active agent(s) tobe delivered by the pharmaceutical formulation. In alternateembodiments, the delayed-release component provides about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% of the total dosage of theactive agent(s) to be delivered by the formulation.

A delayed-release formulation typically comprises a barrier coating thatdelays the release of the active ingredient(s). The barrier coating mayconsist of a variety of different materials, depending on the objective.In addition, a formulation may comprise a plurality of barrier coatingsto facilitate release in a temporal manner. The coating may be a sugarcoating, a film coating (e.g., based on hydroxypropyl methylcellulose,methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose,carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/orpolyvinylpyrrolidone), or a coating based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose. Furthermore, the formulationmay additionally include a time delay material such as, for example,glyceryl monostearate or glyceryl distearate.

In some embodiments, the delayed-release formulation includes an entericcoating comprised one or more polymers facilitating release of activeagents in proximal or distal regions of the gastrointestinal tract. Asused herein, the term “enteric polymer coating” a coating comprising ofone or more polymers having a pH dependent or pH-independent releaseprofile. Typically the coating resists dissolution in the acidic mediumof the stomach, but dissolves or erodes in more distal regions of thegastrointestinal tract, such as the small intestine or colon. An entericpolymer coating typically resists releases of the active agents untilsome time after a gastric emptying lag period of about 3-4 hours afteradministration.

pH dependent enteric coatings comprising one or more pH-dependent orpH-sensitive polymers that maintain their structural integrity at lowpH, as in the stomach, but dissolve in higher pH environments in moredistal regions of the gastrointestinal tract, such as the smallintestine, where the drug contents are released. For purposes of thepresent invention, “pH dependent” is defined as having characteristics(e.g., dissolution) which vary according to environmental pH. ExemplarypH-dependent polymers include, but are not limited to, methacarylic acidcopolymers, methacrylic acid-methyl methacrylate copolymers (e.g.,EUDRAGIT® L100 (Type A), EUDRAGIT® S100 (Type B), Rohm GmbH, Germany;methacrylic acid-ethyl acrylate copolymers (e.g., EUDRAGIT® L100-55(Type C) and EUDRAGIT® L30D-55 copolymer dispersion, Rohm GmbH,Germany); copolymers of methacrylic acid-methyl methacrylate and methylmethacrylate (EUDRAGIT® FS); terpolymers of methacrylic acid,methacrylate, and ethyl acrylate; cellulose acetate phthalates (CAP);hydroxypropyl methylcellulose phthalate (HPMCP) (e.g., HP-55, HP-50,HP-55S, Shinetsu Chemical, Japan); polyvinyl acetate phthalates (PVAP)(e.g., COATERIC®, OPADRY® enteric white OY-P-7171); cellulose acetatesuccinates (CAS); hydroxypropyl methylcellulose acetate succinate(HPMCAS), e.g., HPMCAS LF Grade, MF Grade, HF Grade, including AQOAT® LFand AQOAT® MF (Shin-Etsu Chemical, Japan); Shinetsu Chemical, Japan);shellac (e.g., Marcoat™ 125 & Marcoat™ 125N); carboxymethylethylcellulose (CMEC, Freund Corporation, Japan), cellulose acetatephthalates (CAP)(e.g., AQUATERIC®); cellulose acetate trimellitates(CAT); and mixtures of two or more thereof at weight ratios betweenabout 2:1 to about 5:1, such as, for instance, a mixture of EUDRAGIT® L100-55 and EUDRAGIT® S 100 at a weight ratio of about 3:1 to about 2:1,or a mixture of EUDRAGIT® L 30 D-55 and EUDRAGIT® FS at a weight ratioof about 3:1 to about 5:1.

pH-dependent polymers typically exhibit a characteristic pH optimum fordissolution. In some embodiments, the pH-dependent polymer exhibits a pHoptimum between about 5.0 and 5.5, between about 5.5 and 6.0, betweenabout 6.0 and 6.5, or between about 6.5 and 7.0. In other embodiments,the pH-dependent polymer exhibits a pH optimum of ≧5.0, of ≧5.5, of≧6.0, of ≧6.5, or of ≧7.0.

In other embodiments, the enteric coating may comprise one or morepH-independent polymers. These polymers provide for release of the drugafter a certain time, independent of the pH. For purposes of the presentinvention, “pH independent” is defined as having characteristics (e.g.,dissolution) which are substantially unaffected by pH. pH independentpolymers are often referred to in the context of “time-controlled” or“time-dependent”release profiles.

A pH independent polymer may be water-insoluble or water soluble.Exemplary water insoluble pH independent polymers include, but are notlimited to, neutral methacrylic acid esters with a small portion oftrimethylammonioethyl methacrylate chloride (e.g., EUDRAGIT® RS andEUDRAGIT® RL; neutral ester dispersions without any functional groups(e.g., EUDRAGIT® NE30D and EUDRAGIT® NE30); cellulosic polymers, such asethylcellulose, hydroxyl ethyl cellulose, cellulose acetate or mixturesand other pH independent coating products. Exemplary water soluble pHindependent polymers include OPADRY®amb.

In some embodiments, the pH independent polymers contain one or morepolysaccharides that are resistant to erosion in both the stomach andintestine. Such polymers can be only degraded in the colon, whichcontains a large microflora containing biodegradable enzymes breakingdown, for example, the polysaccharide coatings to release the drugcontents in a controlled, time-dependent manner.

In certain embodiment, the coating methodology employs the blending ofone or more pH-dependent and one or more pH-independent polymers. Theblending of pH-dependent and pH-independent polymers can reduce therelease rate of active ingredients once the soluble polymer has reachedits optimum pH of solubilization.

In some embodiments, a “time-controlled” or “time-dependent” releaseprofile can be obtained using a water insoluble capsule body containingone or more active agents, wherein the capsule body closed at one endwith an insoluble, but permeable and swellable hydrogel plug. Uponcontact with gastrointestinal fluid or dissolution medium, the plugswells, pushing itself out of the capsule and releasing the drugs aftera pre-determined lag time, which can be controlled by e.g., the positionand dimensions of the plug. The capsule body may be further coated withan outer pH-dependent enteric coating keeping the capsule intact untilit reaches the small intestine. Suitable plug materials include, forexample, polymethacrylates, erodible compressed polymers (e.g., HPMC,polyvinyl alcohol), congealed melted polymer (e.g., glyceryl monooleate) and enzymatically controlled erodible polymers (e.g.,polysaccharides, such as amylose, arabinogalactan, chitosan, chondroitinsulfate, cyclodextrin, dextran, guar gum, pectin and xylan).

In other embodiments, capsules or bilayered tablets may be formulated tocontain a drug-containing core, covered by a swelling layer, and anouter insoluble, but semi-permeable polymer coating or membrane. The lagtime prior to rupture can be controlled by the permeation and mechanicalproperties of the polymer coating and the swelling behavior of theswelling layer. Typically, the swelling layer comprises one or moreswelling agents, such as swellable hydrophilic polymers that swell andretain water in their structures.

Exemplary water swellable materials include, but are not limited to,polyethylene oxide (having e.g., an average molecular weight between1,000,000 to 7,000,000, such as POLYOX®), methylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose; polyalkylene oxides having aweight average molecular weight of 100,000 to 6,000,000, including butnot limited to poly(methylene oxide), poly(butylene oxide); poly(hydroxyalkyl methacrylate) having a molecular weight of from 25,000 to5,000,000; poly(vinyl)alcohol, having a low acetal residue, which iscross-linked with glyoxal, formaldehyde or glutaraldehyde and having adegree of polymerization of from 200 to 30,000; mixtures of methylcellulose, cross-linked agar and carboxymethyl cellulose; hydrogelforming copolymers produced by forming a dispersion of a finely dividedcopolymer of maleic anhydride with styrene, ethylene, propylene,butylene or isobutylene cross-linked with from 0.001 to 0.5 moles ofsaturated cross-linking agent per mole of maleic anyhydride in thecopolymer; CARBOPOL® acidic carboxy polymers having a molecular weightof 450,000 to 4,000,000; CYANAMER® polyacrylamides; cross-linked waterswellable indenemaleicanhydride polymers; GOODRITE® polyacrylic acidhaving a molecular weight of 80,000 to 200,000; starch graft copolymers;AQUA-KEEPS® acrylate polymer polysaccharides composed of condensedglucose units such as diester cross-linked polyglucan; carbomers havinga viscosity of 3,000 to 60,000 mPa as a 0.5%-1% w/v aqueous solution;cellulose ethers such as hydroxypropylcellulose having a viscosity ofabout 1000-7000 mPa s as a 1% w/w aqueous solution (25° C.);hydroxypropyl methylcellulose having a viscosity of about 1000 orhigher, preferably 2,500 or higher to a maximum of 25,000 mPa as a 2%w/v aqueous solution; polyvinylpyrrolidone having a viscosity of about300-700 mPa s as a 10% w/v aqueous solution at 20° C.; and combinationsthereof.

The enteric layer may further comprise anti-tackiness agents, such astalc or glyceryl monostearate and/or plasticizers such as. The entericlayer may further comprise one or more plasticizers including, but notlimited to, triethyl citrate, acetyl triethyl citrate, acetyltributylcitrate, polyethylene glycol acetylated monoglycerides, glycerin,triacetin, propylene glycol, phthalate esters (e.g., diethyl phthalate,dibutyl phthalate), titanium dioxide, ferric oxides, castor oil,sorbitol and dibutyl sebacate.

In another embodiment, the delay release formulation employs awater-permeable but insoluble film coating to enclose the activeingredient and an osmotic agent utilizing a enclosing. As water from thegut slowly diffuses through the film into the core, the core swellsuntil the film bursts, thereby releasing the active ingredients. Thefilm coating may be adjusted to permit various rates of water permeationor release time.

Alternatively, the release time of the drugs can be controlled by adisintegration lag time depending on the balance between thetolerability and thickness of a water insoluble polymer membrane (suchas ethyl cellulose, EC) containing predefined micropores at the bottomof the body and the amount of a swellable excipient, such as lowsubstituted hydroxypropyl cellulose (L-HPC) and sodium glycolate. Afteroral administration, GI fluids permeate through the micropores, causingswelling of the swellable excipients, which produces an inner pressuredisengaging the capsular components, including a first capsule bodycontaining the swellable materials, a second capsule body containing thedrugs, and an outer cap attached to the first capsule body.

In other embodiments, the drugs may be released by an osmotic mechanism.By way of example, a capsule may be formulated with a single osmoticunit or it may incorporate 2, 3, 4, 5, or 6 push-pull units encapsulatedwithin a hard gelatin capsule, whereby each bilayer push pull unitcontains an osmotic push layer and a drug layer, both surrounded by asemi-permeable membrane. One or more orifices are drilled through themembrane next to the drug layer. This membrane may be additionallycovered with a pH-dependent enteric coating to prevent release untilafter gastric emptying. The gelatin capsule dissolves immediately afteringestion. As the push pull unit(s) enter the small intestine, theenteric coating breaks down, which then allows fluid to flow through thesemi-permeable membrane, swelling the osmotic push compartment to forcedrugs out through the orifice(s) at a rate precisely controlled by therate of water transport through the semi-permeable membrane. Release ofdrugs can occur over a constant rate for up to 24 hours or more.

The osmotic push layer comprises one or more osmotic agents creating thedriving force for transport of water through the semi-permeable membraneinto the core of the delivery vehicle. One class of osmotic agentsincludes water-swellable hydrophilic polymers, also referred to as“osmopolymers” and “hydrogels,” including, but not limited to,hydrophilic vinyl and acrylic polymers, polysaccharides such as calciumalginate, polyethylene oxide (PEO), polyethylene glycol (PEG),polypropylene glycol (PPG), poly(2-hydroxyethyl methacrylate),poly(acrylic) acid, poly(methacrylic) acid, polyvinylpyrrolidone (PVP),crosslinked PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers, PVA/PVPcopolymers with hydrophobic monomers such as methyl methacrylate andvinyl acetate, hydrophilic polyurethanes containing large PEO blocks,sodium croscarmellose, carrageenan, hydroxyethyl cellulose (HEC),hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC),carboxymethyl cellulose (CMC) and carboxyethyl, cellulose (CEC), sodiumalginate, polycarbophil, gelatin, xanthan gum, and sodium starchglycolate.

Another class of osmotic agents includes osmogens, which are capable ofimbibing water to effect an osmotic pressure gradient across thesemi-permeable membrane. Exemplary osmogens include, but are not limitedto, inorganic salts, such as magnesium sulfate, magnesium chloride,calcium chloride, sodium chloride, lithium chloride, potassium sulfate,potassium phosphates, sodium carbonate, sodium sulfite, lithium sulfate,potassium chloride, and sodium sulfate; sugars, such as dextrose,fructose, glucose, inositol, lactose, maltose, mannitol, raffinose,sorbitol, sucrose, trehalose, and xylitol; organic acids, such asascorbic acid, benzoic acid, fumaric acid, citric acid, maleic acid,sebacic acid, sorbic acid, adipic acid, edetic acid, glutamic acid,p-toluenesulfonic acid, succinic acid, and tartaric acid; urea; andmixtures thereof.

Materials useful in forming the semipermeable membrane include variousgrades of acrylics, vinyls, ethers, polyamides, polyesters, andcellulosic derivatives that are water-permeable and water-insoluble atphysiologically relevant pHs, or are susceptible to being renderedwater-insoluble by chemical alteration, such as crosslinking.

In another embodiment, the delay release formulation employs awater-impermeable tablet coating whereby water enters through acontrolled aperture in the coating until the core bursts. When thetablet bursts, the drug contents are released immediately or over alonger period of time. These and other techniques may be modified toallow for a pre-determined lag period before release of drugs isinitiated.

Various coating techniques may be applied to granules, beads, powders orpellets, tablets, capsules or combinations thereof containing activeagents to produce different and distinct release profiles. In someembodiments, the pharmaceutical composition is in a tablet or capsuleform containing a single coating layer. In other embodiments, thepharmaceutical composition is in a tablet or capsule form containingmultiple coating layers.

In some embodiments, the pharmaceutical composition comprises aplurality of active ingredients selected from the group consisting ofanalgesics, antimuscarinic agents, antidiuretics and spasmolytics.Examples of spasmolytics include, but are not limited to, carisoprodol,benzodiazepines, baclofen, cyclobenzaprine, metaxalone, methocarbamol,clonidine, clonidine analog, and dantrolene. In some embodiments, thepharmaceutical composition comprises one or more analgesics. In otherembodiments, the pharmaceutical composition comprises (1) one or moreanalgesics, and (2) one or more other active ingredients selected fromthe group consisting of antimuscarinic agents, antidiuretics andspasmolytics. In another embodiment, the pharmaceutical compositioncomprises (1) one or two analgesics and (2) one or two antimuscarinicagents. In another embodiment, the pharmaceutical composition comprises(1) one or two analgesics and (2) one or two antidiuretics. In anotherembodiment, the pharmaceutical composition comprises (1) one or twoanalgesics and (2) one or two spasmolytics. In yet another embodiment,the pharmaceutical composition comprises (1) one or two analgesics, (2)one or two antimuscarinic agents, and (3) one or two antidiuretics.

In one embodiment, the plurality of active ingredients are formulatedfor immediate-release. In other embodiment, the plurality of activeingredients are formulated for delayed-release. In other embodiment, theplurality of active ingredients are formulated for bothimmediate-release and delayed-release (e.g., a first portion of eachactive ingredient is formulated for immediate-release and a secondportion of each active ingredient is formulated for delayed-release). Inyet other embodiment, some of the plurality of active ingredients areformulated for immediate-release and some of the plurality of activeingredients are formulated for delayed-release (e.g., active ingredientsA, B, C are formulated for immediate-release and active ingredients Cand D are formulated for delayed-release).

In certain embodiments, the pharmaceutical composition comprises animmediate-release component and a delayed-release component. Theimmediate-release component may comprise one or more active ingredientsselected from the group consisting of analgesics, antimuscarinic agents,antidiuretics and spasmolytics. The delayed-release component maycomprise one or more active ingredients selected from the groupconsisting of analgesics, antimuscarinic agents, antidiuretics andspasmolytics. In some embodiments, the immediate-release component andthe delayed-release component have exactly the same active ingredients.In other embodiments, the immediate-release component and thedelayed-release component have different active ingredients. In yetother embodiments, the immediate-release component and thedelayed-release component have one or more common active ingredients.

In one embodiment, the pharmaceutical composition comprises two activeingredients (e.g., two analgesic agents, or a mixture of one analgesicagent and one antimuscarinic agent or antiuretic or spasmolytic),formulated for immediate-release at about the same time. In anotherembodiment, the pharmaceutical composition comprises two activeingredients, formulated for delayed-release at about the same time. Inanother embodiment, the pharmaceutical composition comprises two activeingredients formulated as two delayed-release components, each providinga different delayed-release profile. For example, a firstdelayed-release component releases a first active ingredient at a firsttime point and a second delayed-release component releases a secondactive ingredient at a second time point. In another embodiment, thepharmaceutical composition comprises two active ingredients, one isformulated for immediate-release and the other is formulated fordelayed-release.

In other embodiments, the pharmaceutical composition comprises twoactive ingredients (e.g., two analgesic agents, or a mixture of oneanalgesic agent and one antimuscarinic agent or antiuretic orspasmolytic) formulated for immediate-release, and (2) two activeingredients (e.g., two analgesic agents, or a mixture of one analgesicagent and one antimuscarinic agent or antiuretic or spasmolytic)formulated for delayed-release. In other embodiments, the pharmaceuticalcomposition comprises three active ingredients formulated forimmediate-release, and (2) three active ingredients formulated fordelayed-release. In other embodiments, the pharmaceutical compositioncomprises four active ingredients formulated for immediate-release, and(2) four active ingredients formulated for delayed-release. In theseembodiments, the active ingredient(s) in the immediate-release componentcan be the same as, or different from, the active ingredient(s) in thedelayed-release component.

The term “immediate-release” is used herein with reference to a drugformulation that does not contain a dissolution rate controllingmaterial. There is substantially no delay in the release of the activeagents following administration of an immediate-release formulation. Animmediate-release coating may include suitable materials immediatelydissolving following administration so as to release the drug contentstherein. Exemplary immediate-release coating materials include gelatin,polyvinyl alcohol polyethylene glycol (PVA-PEG) copolymers (e.g.,KOLLICOAT®) and various others materials known to those skilled in theart.

An immediate-release composition may comprise 100% of the total dosageof a given active agent administered in a single unit dose.Alternatively, an immediate-release component may be included as acomponent in a combined release profile formulation that may provideabout 1% to about 50% of the total dosage of the active agent(s) to bedelivered by the pharmaceutical formulation. For example, theimmediate-release component may provide at least about 5%, or about 10%to about 30%, or about 45% to about 50% of the total dosage of theactive agent(s) to be delivered by the formulation. The rest of theactive agent(s) may be delivered in a delayed-release formulation. Inalternate embodiments, the immediate-release component provides about10, 15, 20, 25, 30, 35, 40, 45 or 50% of the total dosage of the activeagent(s) to be delivered by the formulation. The delayed-releasecomponent provides about 90, 85, 80, 75, 70, 65, 60, 55 or 50% of thetotal dosage of the active agent(s) to be delivered by the formulation.

In some embodiments, the immediate-release or delayed-releaseformulation comprises an active core comprised of one or more inertparticles, each in the form of a bead, pellet, pill, granular particle,microcapsule, microsphere, microgranule, nanocapsule, or nanospherecoated on its surfaces with drugs in the form of e.g., a drug-containingfilm-forming composition using, for example, fluid bed techniques orother methodologies known to those of skill in the art. The inertparticle can be of various sizes, so long as it is large enough toremain poorly dissolved. Alternatively, the active core may be preparedby granulating and milling and/or by extrusion and spheronization of apolymer composition containing the drug substance.

The amount of drug in the core will depend on the dose that is required,and typically varies from about 5 to 90 weight %. Generally, thepolymeric coating on the active core will be from about 1 to 50% basedon the weight of the coated particle, depending on the lag time and typeof release profile required and/or the polymers and coating solventschosen. Those skilled in the art will be able to select an appropriateamount of drug for coating onto or incorporating into the core toachieve the desired dosage. In one embodiment, the inactive core may bea sugar sphere or a buffer crystal or an encapsulated buffer crystalsuch as calcium carbonate, sodium bicarbonate, fumaric acid, tartaricacid, etc. which alters the microenvironment of the drug to facilitateits release.

In some embodiments, the delayed-release formulation is formed bycoating a water soluble/dispersible drug-containing particle, such as abead, with a mixture of a water insoluble polymer and an entericpolymer, wherein the water insoluble polymer and the enteric polymer maybe present at a weight ratio of from 4:1 to 1:1, and the total weight ofthe coatings is 10 to 60 weight % based on the total weight of thecoated beads. The drug layered beads may optionally include an innerdissolution rate controlling membrane of ethylcellulose. The compositionof the outer layer, as well as the individual weights of the inner andouter layers of the polymeric membrane are optimized for achievingdesired circadian rhythm release profiles for a given active, which arepredicted based on in vitro/in vivo correlations.

In other embodiments the formulations comprise a mixture ofimmediate-release drug-containing particles without a dissolution ratecontrolling polymer membrane and delayed-release beads exhibiting, forexample, a lag time of 2-4 hours following oral administration, thusproviding a two-pulse release profile. In yet other embodiments theformulations comprise a mixture of two types of delayed-release beads: afirst type that exhibits a lag time of 1-3 hours and a second type thatexhibits a lag time of 4-6 hours.

In some embodiments, the active core is coated with one or more layersof dissolution rate-controlling polymers to obtain desired releaseprofiles with or without a lag time. An inner layer membrane can largelycontrol the rate of drug release following imbibition of water or bodyfluids into the core, while the outer layer membrane can provide for adesired lag time (the period of no or little drug release followingimbibition of water or body fluids into the core). The inner layermembrane may comprise a water insoluble polymer, or a mixture of waterinsoluble and water soluble polymers.

The polymers suitable for the outer membrane, which largely controls thelag time of up to 6 hours may comprise an enteric polymer, as describedabove, and a water insoluble polymer at 10 to 50 weight %. The ratio ofwater insoluble polymer to enteric polymer may vary from 4:1 to 1:2,preferably the polymers are present at a ratio of about 1:1. The waterinsoluble polymer typically used is ethylcellulose.

Exemplary water insoluble polymers include ethylcellulose, polyvinylacetate (Kollicoat SR#0D from BASF), neutral copolymers based on ethylacrylate and methylmethacrylate, copolymers of acrylic and methacrylicacid esters with quaternary ammonium groups such as EUDRAGIT® NE, RS andRS30D, RL or RL30D and the like. Exemplary water soluble polymersinclude low molecular weight HPMC, HPC, methylcellulose, polyethyleneglycol (PEG of molecular weight>3000) at a thickness ranging from 1weight % up to 10 weight % depending on the solubility of the active inwater and the solvent or latex suspension based coating formulationused. The water insoluble polymer to water soluble polymer may typicallyvary from 95:5 to 60:40, preferably from 80:20 to 65:35.

Preferably, the formulations are designed with release profiles to limitinterference with restful sleep, wherein the formulation releases themedicine when the individual would normally be awakened by an urge tourinate. For example, consider an individual who begins sleeping at 11PM and is normally awakened at 12:30 AM, 3:00 AM, and 6:00 AM tourinate. A delayed-release vehicle could deliver the medicine at 12:15AM, thereby delaying the need to urinate for perhaps 2-3 hours.

The pharmaceutical composition may be administered daily or administeredon an as needed basis. In certain embodiments, the pharmaceuticalcomposition is administered to the subject prior to bedtime. In someembodiments, the pharmaceutical composition is administered immediatelybefore bedtime. In some embodiments, the pharmaceutical composition isadministered within about two hours before bedtime, preferably withinabout one hour before bedtime. In another embodiment, the pharmaceuticalcomposition is administered about two hours before bedtime. In a furtherembodiment, the pharmaceutical composition is administered at least twohours before bedtime. In another embodiment, the pharmaceuticalcomposition is administered about one hour before bedtime. In a furtherembodiment, the pharmaceutical composition is administered at least onehour before bedtime. In a still further embodiment, the pharmaceuticalcomposition is administered less than one hour before bedtime. In stillanother embodiment, the pharmaceutical composition is administeredimmediately before bedtime. Preferably, the pharmaceutical compositionis administered orally.

The appropriate dosage (“therapeutically effective amount”) of theactive agent(s) in the immediate-component or delayed-release componentwill depend, for example, the severity and course of the condition, themode of administration, the bioavailability of the particular agent(s),the age and weight of the patient, the patient's clinical history andresponse to the active agent(s), discretion of the physician, etc.

As a general proposition, the therapeutically effective amount of theactive agent(s) in the immediate-release component or thedelayed-release component is administered in the range of about 100μg/kg body weight/day to about 100 mg/kg body weight/day whether by oneor more administrations. In some embodiments, the range of each activeagent administered daily is from about 100 μg/kg body weight/day toabout 50 mg/kg body weight/day, 100 μg/kg body weight/day to about 10mg/kg body weight/day, 100 μg/kg body weight/day to about 1 mg/kg bodyweight/day, 100 μg/kg body weight/day to about 10 mg/kg body weight/day,500 μg/kg body weight/day to about 100 mg/kg body weight/day, 500 μg/kgbody weight/day to about 50 mg/kg body weight/day, 500 μg/kg bodyweight/day to about 5 mg/kg body weight/day, 1 mg/kg body weight/day toabout 100 mg/kg body weight/day, 1 mg/kg body weight/day to about 50mg/kg body weight/day, 1 mg/kg body weight/day to about 10 mg/kg bodyweight/day, 5 mg/kg body weight/dose to about 100 mg/kg body weight/day,5 mg/kg body weight/dose to about 50 mg/kg body weight/day, 10 mg/kgbody weight/day to about 100 mg/kg body weight/day, and 10 mg/kg bodyweight/day to about 50 mg/kg body weight/day.

The active agent(s) described herein may be included in animmediate-release component or a delayed-release component orcombinations thereof for daily oral administration at a single dose orcombined dose range of 1 mg to 2000 mg, 5 mg to 2000 mg, 10 mg to 2000mg, 50 mg to 2000 mg, 100 mg to 2000 mg, 200 mg to 2000 mg, 500 mg to2000 mg, 5 mg to 1800 mg, 10 mg to 1600 mg, 50 mg to 1600 mg, 100 mg to1500 mg, 150 mg to 1200 mg, 200 mg to 1000 mg, 300 mg to 800 mg, 325 mgto 500 mg, 1 mg to 1000 mg, 1 mg to 500 mg, 1 mg to 200 mg, 5 mg to 1000mg, 5 mg to 500 mg, 5 mg to 200 mg, 10 mg to 1000 mg, 10 mg to 500 mg,10 mg to 200 mg, 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg, 100mg to 250 mg, 100 mg to 500 mg, 250 mg to 1000 mg, 250 mg to 500 mg, 500mg to 1000 mg, 500 mg to 2000 mg. As expected, the dosage will bedependant on the condition, size, age and condition of the patient.

In some embodiments, the pharmaceutical composition comprises a singleanalgesic agent. In one embodiment, the single analgesic agent isaspirin. In another embodiment, the single analgesic agent is ibuprofen.In another embodiment, the single analgesic agent is naproxen sodium. Inanother embodiment, the single analgesic agent is indomethacin. Inanother embodiment, the single analgesic agent is nabumetone. In anotherembodiment, the single analgesic agent is acetaminophen.

In some embodiments, the single analgesic agent is given at a daily doseof 1 mg to 2000 mg, 5 mg to 2000 mg, 20 mg to 2000 mg, 5 mg to 1000 mg,20 mg to 1000 mg, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to 500 mg,250 mg to 1000 mg or 500 mg to 1000 mg. In certain embodiments, thepharmaceutical composition comprises acetylsalicylic acid, ibuprofen,naproxen sodium, indomethancin, nabumetone or acetaminophen as a singleanalgesic agent and the analgesic agent is administered orally at adaily dose in the range of 5 mg to 2000 mg, 20 mg to 2000 mg, 5 mg to1000 mg, 20 mg to 1000 mg, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to500 mg, 250 mg to 1000 mg or 500 mg to 1000 mg. In some embodiments, asecond analgesic agent is given at a daily dose of 1 mg to 2000 mg, 5 mgto 2000 mg, 20 mg to 2000 mg, 5 mg to 1000 mg, 20 mg to 1000 mg, 50 mgto 500 mg, 100 mg to 500 mg, 250 mg to 500 mg, 250 mg to 1000 mg or 500mg to 1000 mg.

In other embodiments, the pharmaceutical composition comprises a pair ofanalgesic agents. Examples of such paired analgesic agents include, butare not limited to, acetylsalicylic acid and ibuprofen, acetylsalicylicacid and naproxen sodium, acetylsalicylic acid and nabumetone,acetylsalicylic acid and acetaminophen, acetylsalicylic acid andindomethancin, ibuprofen and naproxen sodium, ibuprofen and nabumetone,ibuprofen and acetaminophen, ibuprofen and indomethancin, naproxensodium and nabumetone, naproxen sodium and acetaminophen, naproxensodium and indomethancin, nabumetone and acetaminophen, nabumetone andindomethancin, and acetaminophen and indomethancin. The paired analgesicagents are mixed at a weight ratio in the range of 0.1:1 to 10:1, 0.2:1to 5:1 or 0.3:1 to 3:1, with a combined dose in the range of 5 mg to2000 mg, 20 mg to 2000 mg, 100 mg to 2000 mg, 200 mg to 2000 mg, 500 mgto 2000 mg, 5 mg to 1500 mg, 20 mg to 1500 mg, 100 mg to 1500 mg, 200 mgto 1500 mg, 500 mg to 1500 mg, 5 mg to 1000 mg, 20 mg to 1000 mg, 100 mgto 1000 mg, 250 mg to 500 mg, 250 mg to 1000 mg, 250 mg to 1500 mg, 500mg to 1000 mg, 500 mg to 1500 mg, 1000 mg to 1500 mg, and 1000 mg to2000 mg. In one embodiment, the paired analgesic agents are mixed at aweight ratio of 1:1.

In some other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more antimuscarinic agents.Examples of the antimuscarinic agents include, but are not limited to,oxybutynin, solifenacin, darifenacin, fesoterodine, tolterodine,trospium and atropine. The daily dose of antimuscarinic agent is in therange of 0.01 mg to 100 mg, 0.1 mg to 100 mg, 1 mg to 100 mg, 10 mg to100 mg, 0.01 mg to 25 mg, 0.1 mg to 25 mg, 1 mg to 25 mg, 10 mg to 25mg, 0.01 mg to 10 mg, 0.1 mg to 10 mg, 1 mg to 10 mg, 10 mg to 100 mgand 10 mg to 25 mg.

Another aspect of the present application relates to a method forreducing the frequency of urination by administering to a person in needthereof a pharmaceutical composition formulated in an immediate-releaseformulation. In some embodiments, the pharmaceutical compositioncomprises one or more analgesic agents and one or more antimuscarinicagents. In other embodiments, the pharmaceutical composition comprisesone or more analgesic agents and one or more antidiuretic agents. In yetother embodiments, the pharmaceutical composition comprises one or moreanalgesic agents, one or more antimuscarinic agents, and one or moreantidiuretic agents.

In other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more spasmolytics. Examples ofspasmolytics include, but are not limited to, carisoprodol,benzodiazepines, baclofen, cyclobenzaprine, metaxalone, methocarbamol,clonidine, clonidine analog, and dantrolene. In some embodiments, thespasmolytics is used at a daily dose of 1 mg to 1000 mg, 1 mg to 100 mg,10 mg to 1000 mg, 10 mg to 100 mg, 20 mg to 1000 mg, 20 mg to 800 mg, 20mg to 500 mg, 20 mg to 200 mg, 50 mg to 1000 mg, 50 mg to 800 mg, 50 mgto 200 mg, 100 mg to 800 mg, 100 mg to 500 mg, 200 mg to 800 mg, and 200mg to 500 mg. The spasmolytics may be formulated, alone or together withother active ingredient(s) in the pharmaceutical composition, forimmediate-release, delayed-extended release or combinations thereof.

In certain embodiments, the pharmaceutical composition comprises two ormore analgesic agents. In other embodiments, the pharmaceuticalcomposition comprises one or more analgesic agents and one or moreantimuscarinic agents and/or antidiuretic agents. The pharmaceuticalcomposition may be formulated into a tablet, capsule, dragee, powder,granulate, liquid, gel or emulsion form. Said liquid, gel or emulsionmay be ingested by the subject in naked form or contained within acapsule.

In certain embodiments, the analgesic agent is selected from the groupconsisting of salicylates, aspirin, salicylic acid, methyl salicylate,diflunisal, salsalate, olsalazine, sulfasalazine, para-aminophenolderivatives, acetanilide, acetaminophen, phenacetin, fenamates,mefenamic acid, meclofenamate, sodium meclofenamate, heteroaryl aceticacid derivatives, tolmetin, ketorolac, diclofenac, propionic acidderivatives, ibuprofen, naproxen sodium, naproxen, fenoprofen,ketoprofen, flurbiprofen, oxaprozin; enolic acids, oxicam derivatives,piroxicam, meloxicam, tenoxicam, ampiroxicam, droxicam, pivoxicam,pyrazolon derivatives, phenylbutazone, oxyphenbutazone, antipyrine,aminopyrine, dipyrone, coxibs, celecoxib, rofecoxib, nabumetone,apazone, nimesulide, indomethacin, sulindac, etodolac, andisobutylphenyl propionic acid. The antimuscarinic agent is selected fromthe group consisting of oxybutynin, solifenacin, darifenacin andatropine.

In some embodiments, the pharmaceutical composition comprises a singleanalgesic agent and a single antimuscarinic agent. In one embodiment,the single analgesic agent is aspirin. In another embodiment, the singleanalgesic agent is ibuprofen. In another embodiment, the singleanalgesic agent is naproxen sodium. In another embodiment, the singleanalgesic agent is indomethacin. In another embodiment, the singleanalgesic agent is nabumetone. In another embodiment, the singleanalgesic agent is acetaminophen. The analgesic agent andanti-muscarinic agent may be given at doses in the ranges describedabove.

Another aspect of the present application relates to a method fortreating nocturia by administering to a subject in need thereof (1) oneor more analgesic agents and (2) one or more antidiuretic agents. Incertain embodiments, the antidiuretic agent(s) act to: (1) increasevasopressin secretion; (2) increase vasopressin receptor activation; (3)reduce secretion of atrial natriuretic peptide (ANP) or C-typenatriuretic peptide (CNP); or (4) reduce ANP and/or CNP receptoractivation.

Exemplary antidiuretic agents include, but are not limited to,antidiuretic hormone (ADH), angiotensin II, aldosterone, vasopressin,vasopressin analogs (e.g., desmopressin argipressin, lypressin,felypressin, ornipressin, terlipressin); vasopressin receptor agonists,atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP)receptor (i.e., NPR1, NPR2, NPR3) antagonists (e.g., HS-142-1, isatin,[Asu7,23′]b-ANP-(7-28)], anantin, a cyclic peptide from Streptomycescoerulescens, and 3G12 monoclonal antibody); somatostatin type 2receptor antagonists (e.g., somatostatin), andpharmaceutically-acceptable derivatives, analogs, salts, hydrates, andsolvates thereof.

In certain embodiments, the one or more analgesic agent and one or moreantidiuretic agents are formulated for delayed-release.

Another aspect of the present application relates to a method fortreating nocturia by administering to a person in need thereof a firstpharmaceutical composition comprising a diuretic, followed with a secondpharmaceutical composition comprising one or more analgesic agents. Thefirst pharmaceutical composition is dosed and formulated to have adiuretic effect within 6 hours of administration and is administered atleast 8 hours prior to bedtime. The second pharmaceutical composition isadministered within 2 hours prior to bedtime.

Examples of diuretics include, but are not limited to, acidifying salts,such as CaCl₂ and NH₄Cl; arginine vasopressin receptor 2 antagonists,such as amphotericin B and lithium citrate; aquaretics, such asGoldenrod and Junipe; Na—H exchanger antagonists, such as dopamine;carbonic anhydrase inhibitors, such as acetazolamide and dorzolamide;loop diuretics, such as bumetanide, ethacrynic acid, furosemide andtorsemide; osmotic diuretics, such as glucose and mannitol;potassium-sparing diuretics, such as amiloride, spironolactone,triamterene, potassium canrenoate; thiazides, such asbendroflumethiazide and hydrochlorothiazide; and xanthines, such ascaffeine, theophylline and theobromine.

In some embodiments, the second pharmaceutical composition furthercomprises one or more antimuscarinic agents. Examples of theantimuscarinic agents include, but are not limited to, oxybutynin,solifenacin, darifenacin, fesoterodine, tolterodine, trospium andatropine. The second pharmaceutical composition may be formulated inimmediate-release formulation or delayed-release formulation. In oneembodiment, the first pharmaceutical composition is formulated forimmediate-release and the second pharmaceutical composition isformulated for delayed-release.

In some other embodiments, the second pharmaceutical composition furthercomprises one or more antidiuretic agents.

Another aspect of the present application relates to a pharmaceuticalcomposition comprising a plurality of active ingredients and apharmaceutically acceptable carrier. In some embodiments, the pluralityof active ingredients comprise two or more analgesics. In otherembodiments, the plurality of active ingredients comprise one or moreanalgesics and one or more antimuscarinic agents. In other embodiments,the plurality of active ingredients comprise one or more analgesics andone or more antidiuretic agents. In yet other embodiments, the pluralityof active ingredients comprise one or more analgesics, one or moreantidiuretic agents, and one or more antimuscarinic agents. In otherembodiments, at least one of said plurality of active ingredients isformulated for delayed-release.

In some embodiments, the pharmaceutical composition comprises twoanalgesics selected from the group consisting of cetylsalicylic acid,ibuprofen, naproxen sodium, nabumetone, acetaminophen and indomethancin.In other embodiments, the pharmaceutical composition comprises one ormore analgesics selected from the group consisting of cetylsalicylicacid, ibuprofen, naproxen sodium, nabumetone, acetaminophen andindomethancin; and an antimuscarinic agent selected from the groupconsisting of oxybutynin, solifenacin, darifenacin and atropine.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, sweeteners and thelike. The pharmaceutically acceptable carriers may be prepared from awide range of materials including, but not limited to, flavoring agents,sweetening agents and miscellaneous materials such as buffers andabsorbents that may be needed in order to prepare a particulartherapeutic composition. The use of such media and agents withpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated.

Another aspect of the present application relates to a method forreducing the frequency of urination by administering to a subject inneed thereof, two or more analgesic agents alternatively to prevent thedevelopment of drug resistance. In one embodiment, the method comprisesadministering a first analgesic agent for a first period of time andthen administering a second analgesic agent for a second period of time.In another embodiment, the method further comprises administering athird analgesic agent for a third period of time. The first, second andthird analgesic agents are different from each other and may be selectedfrom the group consisting of: salicylates, aspirin, salicylic acid,methyl salicylate, diflunisal, salsalate, olsalazine, sulfasalazine,para-aminophenol derivatives, acetanilide, acetaminophen, phenacetin,fenamates, mefenamic acid, meclofenamate, sodium meclofenamate,heteroaryl acetic acid derivatives, tolmetin, ketorolac, diclofenac,propionic acid derivatives, ibuprofen, naproxen sodium, naproxen,fenoprofen, ketoprofen, flurbiprofen, oxaprozin; enolic acids, oxicamderivatives, piroxicam, meloxicam, tenoxicam, ampiroxicam, droxicam,pivoxicam, pyrazolon derivatives, phenylbutazone, oxyphenbutazone,antipyrine, aminopyrine, dipyrone, coxibs, celecoxib, rofecoxib,nabumetone, apazone, nimesulide, indomethacin, sulindac, etodolac, andisobutylphenyl propionic acid.

In one embodiment, the first analgesic agent is acetaminophen, thesecond analgesic agent is ibuprofen and the third analgesic agent isnaproxen sodium. The length of each period may vary depending on thesubject's response to each analgesic agent. In some embodiments, eachperiod lasts from 3 days to three weeks. In another embodiment, one ormore of the first, second and third analgesic is formulated fordelayed-release.

The present invention is further illustrated by the following examplewhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

Example 1 Inhibition of the Urge to Urinate

Twenty volunteer subjects, both male and female were enrolled, each ofwhich experienced premature urge or desire to urinate, interfering withtheir ability to sleep for a sufficient period of time to feeladequately rested. Each subject ingested 400-800 mg of ibuprofen as asingle dose prior to bedtime. At least 14 subjects reported that theywere able to rest better because they were not being awakened asfrequently by the urge to urinate.

Several subjects reported that after several weeks of nightly use ofibuprofen, the benefit of less frequent urges to urinate was no longerbeing realized. However, all of these subjects further reported thereturn of the benefit after several days of abstaining from taking thedosages.

Example 2 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Macrophage Responses to Inflammatory andNon-Inflammatory Stimuli Experimental Design

This study is designed to determine the dose and in vitro efficacy ofanalgesics and antimuscarinic agents in controlling macrophage responseto inflammatory and non-inflammatory stimuli mediated by Cox-2 andprostaglandins (PGE, PGH, etc.). It establishes baseline (dose andkinetic) responses to inflammatory and non-inflammatory effectors inbladder cells. Briefly, cultured cells are exposed to analgesic agentsand/or antimuscarinic agents in the absence or presence of variouseffectors.

The effectors include: lipopolysaccharide (LPS), an inflammatory agentand COX2 inducer, as inflammatory stimuli; carbachol or acetylcholine, astimulator of smooth muscle contraction, as non-inflammatory stimuli;botulinum neurotoxin A, a known inhibitor of acetylcholine release, aspositive control; and arachidonic acid (AA), gamma linolenic acid (DGLA)or eicosapentaenoic acid (EPA) as precursors of prostaglandins, whichare produced following the sequential oxidation of AA, DGLA or EPAinside the cell by cyclooxygenases (COX1 and COX2) and terminalprostaglandin synthases.

The analgesic agents include: Salicylates such as aspirin,iso-butyl-propanoic-phenolic acid derivative (ibuprofen) such as Advil,Motrin, Nuprin, and Medipren, naproxen sodium such as Aleve, Anaprox,Antalgin, Feminax Ultra, Flanax, Inza, Midol Extended Relief, Nalgesin,Naposin, Naprelan, Naprogesic, Naprosyn, Naprosyn suspension,EC-Naprosyn, Narocin, Proxen, Synflex and Xenobid, acetic acidderivative such as indomethacin (Indocin),1-naphthaleneacetic acidderivative such as nabumetone or relafen, N-acetyl-para-aminophenol(APAP) derivative such as acetaminophen or paracetamol (Tylenol) andCelecoxib.

The antimuscarinic agents include: oxybutynin, solifenacin, darifenacinand atropine.

Macrophages are subjected to short term (1-2 hrs) or long term (24-48hrs) stimulation of with:

-   -   1) Each analgesic agent alone at various doses.    -   (2) Each analgesic agent at various doses in the presence of        LPS.    -   (3) Each analgesic agent at various doses in the presence of        carbachol or acetylcholine.    -   (4) Each analgesic agent at various doses in the presence of AA,        DGLA, or EPA.    -   (5) Botulinum neurotoxin A alone at various doses.    -   (6) Botulinum neurotoxin A at various doses in the presence of        LPS.    -   (7) Botulinum neurotoxin A at various doses in the presence of        carbachol or acetylcholine.    -   (8) Botulinum neurotoxin A at various doses in the presence of        AA, DGLA, or EPA.    -   (9) Each antimuscarinic agent alone at various doses.    -   (10) Each antimuscarinic agent at various doses in the presence        of LPS.    -   (11) Each antimuscarinic agent at various doses in the presence        of carbachol or acetylcholine.    -   (12) Each antimuscarinic agent at various doses in the presence        of AA, DGLA, or EPA.

The cells are then analyzed for the release of PGH₂, PGE, PGE₂,Prostacydin, Thromboxane, IL-1β, IL-6, TNF-α, the COX2 activity, theproduction of cAMP and cGMP, the production of IL-1β, IL-6, TNF-α andCOX2 mRNA, and surface expression of CD80, CD86 and MHC class IImolecules.

Materials and Methods Macrophage Cells

Murine RAW264.7 or J774 macrophage cells (obtained from ATCC) were usedin this study. Cells were maintained in a culture medium containing RPMI1640 supplemented with 10% fetal bovine serum (FBS), 15 mM HEPES, 2 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml of streptomycin. Cellswere cultured at 37° C. in a 5% CO₂ atmosphere and split (passages) oncea week.

In Vitro Treatment of Macrophage Cells with Analgesics

RAW264.7 macrophage cells were seeded in 96-well plates at a celldensity of 1.5×10⁵ cells per well in 100 μl of the culture medium. Thecells were treated with (1) various concentrations of analgesic(acetaminophen, aspirin, ibuprophen or naproxen), (2) variousconcentrations of lipopolysaccharide (LPS), which is an effector ofinflammatory stimuli to macrophage cells, (3) various concentrations ofcarbachol or acetylcholine, which are effectors of non-inflammatorystimuli, (4) analgesic and LPS or (5) analgesic and carbachol oracetylcholine. Briefly, the analgesics were dissolved in FBS-freeculture medium (i.e., RPMI 1640 supplemented with 15 mM HEPES, 2 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml of streptomycin), anddiluted to desired concentrations by serial dilution with the samemedium. For cells treated with analgesic in the absence of LPS, 50 μl ofanalgesic solution and 50 μl of FBS-free culture medium were added toeach well. For cells treated with analgesic in the presence of LPS, 50μl of analgesic solution and 50 μl of LPS (from Salmonella typhimurium)in FBS-free culture medium were added to each well. All conditions weretested in duplicates.

After 24 or 48 hours of culture, 150 μl of culture supernatants werecollected, spun down for 2 min at 8,000 rpm at 4° C. to remove cells anddebris and stored at −70° C. for analysis of cytokine responses byELISA. The cells were collected and washed by centrifugation (5 min at1,500 rpm at 4° C.) in 500 μl of Phosphate buffer (PBS). Half of thecells were then snap frozen in liquid nitrogen and stored at −70° C. Theremaining cells were stained with fluorescent monoclonal antibodies andanalyzed by flow cytometry.

Flow Cytometry Analysis of Co-Stimulatory Molecule Expression

For flow cytometry analysis, macrophages were diluted in 100 μl of FACSbuffer (phosphate buffered saline (PBS) with 2% bovine serum albumin(BSA) and 0.01% NaN₃) and stained 30 min at 4° C. by addition ofFITC-conjugated anti-CD40, PE-conjugated anti-CD80, PE-conjugatedanti-CD86 antibody, anti MHC class II (I-A^(d)) PE (BD Bioscience).Cells were then washed by centrifugation (5 min at 1,500 rpm at 4° C.)in 300 μl of FACS buffer. After a second wash, cells were re-suspendedin 200 μl of FACS buffer and the percentage of cells expressing a givenmarker (single positive), or a combination of markers (double positive)were analyzed with the aid of an Accuri C6 flow cytometer (BDBiosciences).

Analysis of Cytokine Responses by ELISA

Culture supernatants were subjected to cytokine-specific ELISA todetermine IL-1β, IL-6 and TNF-α responses in cultures of macrophagestreated with analgesic, LPS alone or a combination of LPS and analgesic.The assays were performed on Nunc MaxiSorp Immunoplates (Nunc) coatedovernight with 100 μl of anti-mouse IL-6, TNF-α mAbs (BD Biosciences) orIL-1β mAb (R&D Systems) in 0.1 M sodium bicarbonate buffer (pH 9.5).After two washes with PBS (200 μl per well), 200 μl of PBS 3% BSA wereadded in each well (blocking) and the plates incubated for 2 hours atroom temperature. Plates were washed again two times by addition of 200μl per well, 100 μl of cytokine standards and serial dilutions ofculture supernatants were added in duplicate and the plates wereincubated overnight at 4° C. Finally, the plates were washed twice andincubated with 100 μl of secondary biotinylated anti-mouse IL-6, TNFαmAbs (BD Biosciences) or IL-1β (R&D Systems) followed byperoxidase-labelled goat anti-biotin mAb (Vector Laboratories). Thecolorimetric reaction was developed by the addition of 2,2′-azino-bis(3)-ethylbenzylthiazoline-6-sulfonic acid (ABTS) substrate and H₂O₂(Sigma) and the absorbance measured at 415 nm with a Victor® Vmultilabel plate reader (PerkinElmer).

Determination of COX2 Activity and the Production of cAMP and cGMP

The COX2 activity in the cultured macrophages is determined by COX2activity assay. The production of cAMP and cGMP is determined by thecAMP assay and cGMP assay. These assays are performed routinely in theart.

Results

Table 1 summarizes the experiments performed with Raw 264 macrophagecell line and main findings in terms of the effects of analgesics oncell surface expression of costimulatory molecules CD40 and CD80.Expression of these molecules is stimulated by COX2 and inflammatorysignals and thus, was evaluated to determine functional consequences ofinhibition of COX2.

As shown in Table 2, acetaminophen, aspirin, ibuprophen and naproxeninhibit basal expression of co-stimulatory molecules CD40 and CD80 bymacrophages at all the tested doses (i.e., 5×10⁵ nM, 5×10⁴ nM, 5×10³ nM,5×10² nM, 50 nM and 5 nM), except for the highest dose (i.e., 5×10⁶ nM),which appears to enhance, rather than inhibit, expression of theco-stimulatory molecules. As shown in FIGS. 1A and 1B, such inhibitoryeffect on CD40 and CD50 expression was observed at analgesic doses aslow as 0.05 nM (i.e., 0.00005 μM). This finding supports the notion thata controlled release of small doses of analgesic may be preferable toacute delivery of large doses. The experiment also revealed thatacetaminophen, aspirin, ibuprophen and naproxen have a similarinhibitory effect on LPS induced expression of CD40 and CD80.

TABLE 1 Summary of experiments LPS Salmonella Control typhimuriumAcetaminophen Aspirin Ibuprophen Naproxen TESTS 1 X 2 X Dose responses(0, 5, 50, 1000) ng/mL 3 X Dose responses (0, 5, 50, 500, 5 × 10³, 5 ×10⁴, 5 × 10⁵, 5 × 10⁶) nM 4 X X (5 ng/mL) Dose responses X (50 ng/mL (0,5 , 50, 500, 5 · 10³, 5 · 10⁴, 5 · 10⁵, 5 · 10⁶) nM X (1000 ng/mL)ANALYSIS a Characterization of activation/stimulatory status: Flowcytometry analysis of CD40, CD80, CD86 and MHC class II b Mediators ofinflammatory responses: ELISA analysis of IL-1β, IL-6, TNF-α

TABLE 2 Summary of main findings Negative LPS Effectors % PositiveControl 5 ng/ml 5 · 10⁶ 5 · 10⁵ 5 · 10⁴ 5 · 10³ 500 50 5 CD40⁺CD80⁺ 20.677.8 Dose analgesic (nM) Acetaminophen CD40⁺CD80⁺ 63 18 12 9.8 8.3 9.57.5 Aspirin CD40⁺CD80⁺ 44 11 10.3 8.3 8 10.5 7.5 Ibuprophen CD40⁺CD80⁺ ND* 6.4 7.7 7.9 6.0 4.9 5.8 Naproxen CD40⁺CD80⁺ 37 9.6 7.7 6.9 7.2 6.85.2 Analgesic plus LPS Acetaminophen CD40⁺CD80⁺ 95.1 82.7 72.4 68.8 66.866.2 62.1 Aspirin CD40⁺CD80⁺ 84.5 80 78.7 74.7 75.8 70.1 65.7 IbuprophenCD40⁺CD80⁺ ND 67 77.9 72.9 71.1 63.7 60.3 Naproxen CD40⁺CD80⁺ 66.0 74.177.1 71.0 68.8 72 73 *ND: not done (toxicity)

Table 3 summarizes the results of several studies that measured serumlevels of analgesic after oral therapeutic doses in adult humans. Asshown in Table 3, the maximum serum levels of analgesic after an oraltherapeutic dose are in the range of 10⁴ to 10⁵ nM. Therefore, the dosesof analgesic tested in vitro in Table 2 cover the range ofconcentrations achievable in vivo in humans.

TABLE 3 Serum levels of analgesic in human blood after oral therapeuticdoses Maximum serum levels after oral Molecular therapeutic dosesAnalgesic drug weight mg/L nM References Acetaminophen 151.16 11-18 7.2× 10⁴- BMC Clinical Pharmacology.2010, 10:10 (Tylenol) 1.19 × 10⁵Anaesth Intensive Care. 2011, 39:242 Aspirin 181.66  30-100 1.65 × 10⁵-Disposition of Toxic Drugs and Chemicals in (Acetylsalicylic acid) 5.5 ×10⁵ Man, 8th Edition, Biomedical Public, Foster City, CA, 2008, pp.22-25 J Lab Clin Med. 1984 Jun; 103: 869 Ibuprofen 206.29 24-32 1.16 ×10⁵- BMC Clinical Pharmacology2010, 10: 10 (Advil, Motrin) 1.55 × 10⁵ JClin Pharmacol. 2001, 41: 330 Naproxen 230.26 Up to Up to J ClinPharmacol. 2001, 41: 330 (Aleve) 60 2.6 × 10⁵

Example 3 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Bladder Smooth Muscle Cell Responses toInflammatory and Non-Inflammatory Stimuli Experimental Design

This study is designed to characterize how the optimal doses ofanalgesic determined in Example 2 affect bladder smooth muscle cells incell culture or tissue cultures, and to address whether differentclasses of analgesics can synergize to more efficiently inhibit COX2 andPGE2 responses.

The effectors, analgesic agents and antimuscarinic agents are describedin Example 2.

Primary culture of mouse bladder smooth muscle cells are subjected toshort term (1-2 hrs) or long term (24-48 hrs) stimulation of with:

(1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

The cells are then analyzed for the release of PGH₂, PGE, PGE₂,Prostacydin, Thromboxane, IL-1β, IL-6, TNF-α, the COX2 activity, theproduction of cAMP and cGMP, the production of IL-1β, IL-6, TNF-α andCOX2 mRNA, and surface expression of CD80, CD86 and MHC class IImolecules.

Materials and Methods Isolation and Purification of Murine Bladder Cells

Bladder cells were removed from euthanized animals C57BL/6 mice (8-12weeks old) and cells were isolated by enzymatic digestion followed bypurification on a Percoll gradient. Briefly, bladders from 10 mice wereminced with scissors to fine slurry in 10 ml of digestion buffer (RPMI1640, 2% fetal bovine serum, 0.5 mg/ml collagenase, 30 μg/ml DNase).Bladder slurries were enzymatically digested for 30 minutes at 37° C.Undigested fragments were further dispersed through a cell-trainer. Thecell suspension was pelleted and added to a discontinue 20%, 40% and 75%Percoll gradient for purification on mononuclear cells. Each experimentused 50-60 bladders.

After washes in RPMI 1640, bladder cells were resuspended RPMI 1640supplemented with 10% fetal bovine serum, 15 mM HEPES, 2 mM L-glutamine,100 U/ml penicillin, and 100 μg/ml of streptomycin and seeded inclear-bottom black 96-well cell culture microculture plates at a celldensity of 3×10⁴ cells per well in 100 μl. Cells were cultured at 37° C.in a 5% CO₂ atmosphere.

In Vitro Treatment of Cells with Analgesics

Bladder cells were treated with analgesic solutions (50 μl/well) eitheralone or together with carbachol (10-Molar, 50 μl/well), as an exampleof non-inflammatory stimuli, or lipopolysaccharide (LPS) of Salmonellatyphimurium (1 μg/ml, 50 μl/well), as an example of non-inflammatorystimuli. When no other effectors were added to the cells, 50 μl of RPMI1640 without fetal bovine serum were added to the wells to adjust thefinal volume to 200 μl.

After 24 hours of culture, 150 μl of culture supernatants werecollected, spun down for 2 min at 8,000 rpm at 4° C. to remove cells anddebris and stored at −70° C. for analysis of Prostaglandin E2 (PGE₂)responses by ELISA. Cells were fixed, permeabilized and blocked fordetection of Cyclooxygenase-2 (COX2) using a fluorogenic substrate. Inselected experiment cells were stimulated 12 hours in vitro for analysisof COX2 responses.

Analysis of COX2 Responses

COX2 responses were analyzed by a Cell-Based ELISA using Human/mousetotal COX2 immunoassay (R&D Systems), following the instructions of themanufacturer. Briefly, after cells fixation and permeabilization, amouse anti-total COX2 and a rabbit anti-total GAPDH were added to thewells of the clear-bottom black 96-well cell culture microcultureplates. After incubation and washes, an HRP-conjugated anti-mouse IgGand an AP-conjugated anti-rabbit IgG were added to the wells. Followinganother incubation and set of washes, the HRP- and AP-fluorogenicsubstrates were added. Finally, a Victor® V multilabel plate reader(PerkinElmer) was used to read the fluorescence emitted at 600 nm (COX2fluorescence) and 450 nm (GAPDH fluorescence). Results are expressed asrelative levels of total COX2 as determined by relative fluorescenceunit (RFUs) and normalized to the housekeeping protein GAPDH.

Analysis of PGE2 Responses

Prostaglandin E2 responses were analyzed by a sequential competitiveELISA (R&D Systems). More specifically, culture supernatants or PGE2standards were added to the wells of a 96-well polystyrene microplatecoated with a goat anti-mouse polyclonal antibody. After one hourincubation on a microplate shaker, an HRP-conjugated PGE2 was added andplates incubated for an additional two hours at room temperature. Theplates were then washed and HRP substrate solution added to each well.The color was allowed to develop for 30 min and the reaction stopped bythe addition of sulfuric acid before reading the plate at 450 nm withwavelength correction at 570 nm. Results are expressed as mean pg/ml ofPGE2.

Other Assays

The release of PGH₂, PGE, Prostacydin, Thromboxane, IL-1β, IL-6, andTNF-α, the production of cAMP and cGMP, the production of IL-1β, IL-6,TNF-α and COX2 mRNA, and surface expression of CD80, CD86 and MHC classII molecules are determined as described in Example 2.

Results Analgesics Inhibit COX2 Responses of Murine Bladder Cells to anInflammatory Stimuli

Several analgesics (acetaminophen, aspirin, ibuprofen and naproxen) weretested on mouse bladder cells at the concentration of 5 μM or 50 μM todetermine whether the analgesics could induce COX2 responses. Analysisof 24-hour cultures showed that none of the analgesics tested inducedCOX2 responses in murine bladder cells in vitro.

The effect of these analgesics on the COX2 responses of murine bladdercells to carbachol or LPS stimulation in vitro was also tested. Asindicated in Table 1, the dose of carbachol tested has no significanteffect on COX2 levels in murine bladder cells. On the other hand, LPSsignificantly increased total COX2 levels. Interestingly, acetaminophen,aspirin, ibuprofen and naproxen could all suppress the effect of LPS onCOX2 levels. The suppressive effect of the analgesic was seen when thesedrugs were tested at either 5 μM or 50 μM (Table 4).

TABLE 4 COX2 expression by murine bladder cells after in vitrostimulation and treatment with analgesics Total COX2 levels StimuliAnalgesic (Normalized RFUs) None None 158 ± 18 Carbachol (mM) None 149 ±21 LPS (1 μg/ml) None 420 ± 26 LPS (1 μg/ml) Acetaminophen (5 μM) 275 ±12 LPS (1 μg/ml) Aspirin (5 μM) 240 ± 17 LPS (1 μg/ml) Ibuprofen (5 μM))253 ± 32 LPS (1 μg/ml) Naproxen (5 μM) 284 ± 11 LPS (1 μg/ml)Acetaminophen (50 μM) 243 ± 15 LPS (1 μg/ml) Aspirin (50 μM) 258 ± 21LPS (1 μg/ml) Ibuprofen (50 μM) 266 ± 19 LPS (1 μg/ml) Naproxen (50 μM)279 ± 23

Analgesics Inhibit PGE2 Responses of Murine Bladder Cells to anInflammatory Stimuli

The secretion of PGE2 in culture supernatants of murine bladder cellswas measured to determine the biological significance of the alterationof murine bladder cell COX2 levels by analgesics. As shown in Table 5,PGE2 was not detected in the culture supernatants of unstimulatedbladder cells or bladder cells cultured in the presence of carbachol.Consistent with COX2 responses described above, stimulation of murinebladder cells with LPS induced the secretion of high levels of PGE2.Addition of the analgesics acetaminophen, aspirin, ibuprofen andnaproxen suppressed the effect of LPS on PGE2 secretion and nodifference was seen between the responses of cells treated with the 5 or50 μM dose of analgesic.

TABLE 5 PGE2 secretion by murine bladder cells after in vitrostimulation and treatment with analgesics Stimuli Analgesic PGE2 levels(pg/ml) None None <20.5 Carbachol (mM) None <20.5 LPS (1 μg/ml) None 925± 55 LPS (1 μg/ml) Acetaminophen (5 μM) 619 ± 32 LPS (1 μg/ml) Aspirin(5 μM) 588 ± 21 LPS (1 μg/ml) Ibuprofen (5 μM)) 593 ± 46 LPS (1 μg/ml)Naproxen (5 μM) 597 ± 19 LPS (1 μg/ml) Acetaminophen (50 μM) 600 ± 45LPS (1 μg/ml) Aspirin (50 μM) 571 ± 53 LPS (1 μg/ml) Ibuprofen (50 μM)568 ± 32 LPS (1 μg/ml) Naproxen (50 μM) 588 ± 37

In summary, these data show that the analgesics alone at 5 μM or 50 μMdo not induce COX2 and PGE2 responses in murine bladder cells. Theanalgesics at 5 μM or 50 μM, however, significantly inhibit COX2 andPGE2 responses of murine bladder cells stimulated in vitro with LPS (1μg/ml). No significant effect of analgesics was observed on COX2 andPGE2 responses of murine bladder cells stimulated with carbachol (1 mM).

Example 4 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Bladder Smooth Muscle Cell ContractionExperimental Design

Cultured mouse or rat bladder smooth muscle cells and mouse or ratbladder smooth muscle tissue are exposed to inflammatory stimuli andnon-inflammatory stimuli in the presence of analgesic agent and/orantimuscarinic agent at various concentrations. The stimuli-inducedmuscle contraction is measured to evaluate the inhibitory effect of theanalgesic agent and/or antimuscarinic agent.

The effectors, analgesic agents and antimuscarinic agents are describedin Example 2.

Primary culture of mouse bladder smooth muscle cells are subjected toshort term (1-2 hrs) or long term (24-48 hrs) stimulation of with:

(1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

Materials and Methods

Primary mouse bladder cells are isolated as described in Example 3. Inselected experiments, cultures of bladder tissue are used. Bladdersmooth muscle cell contractions are recorded with a Grass polygraph(Quincy Mass, USA).

Example 5 Effect of Oral Analgesic Agents and Antimuscarinic Agents onCOX2 and PGE2 Responses of Bladder Smooth Muscle Cells ExperimentalDesign:

Normal mice and mice with over active bladder syndrome are given oraldoses of aspirin, naproxen sodium, Ibuprofen, Indocin, nabumetone,Tylenol, Celecoxib, oxybutynin, solifenacin, darifenacin, atropine andcombinations thereof. Control groups include untreated normal mice anduntreated OAB mice without over active bladder syndrome. Thirty (30) minafter the last doses, the bladders are collected and stimulated ex vivowith carbachol or acetylcholine. In selected experiments the bladdersare treated with botulinum neurotoxin A before stimulation withcarbachol. Animals are maintained in metabolic cages and frequency (andvolume) of urination are evaluated. Bladder outputs are determined bymonitoring water intake and cage litter weight. Serum PGH₂, PGE, PGE₂,Prostacydin, Thromboxane, IL-1β, IL-6, TNF-α, cAMP, and cGMP levels aredetermined by ELISA. CD80, CD86, MHC class II expression in whole bloodcells are determined by flow cytometry.

At the end of the experiment, animals are euthanized and ex vivo bladdercontractions are recorded with a Grass polygraph. Portions of bladdersare fixed in formalin, and COX2 responses are analyzed byimmunohistochemistry.

Example 6 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Human Bladder Smooth Muscle Cell Responses toInflammatory and Non-Inflammatory Stimuli Experimental Design

This study is designed to characterize how the optimal doses ofanalgesics determined in Examples 1-5 affect human bladder smooth musclecells in cell culture or tissue cultures, and to address whetherdifferent classes of analgesics can synergize to more efficientlyinhibit COX2 and PGE2 responses.

The effectors, analgesic agents and antimuscarinic agents are describedin Example 2.

Human bladder smooth muscle cells are subjected to short term (1-2 hrs)or long term (24-48 hrs) stimulation of with:

(1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

The cells are then analyzed for the release of PGH₂, PGE, PGE₂,Prostacydin, Thromboxane, IL-1β, IL-6, TNF-α, the COX2 activity, theproduction of cAMP and cGMP, the production of IL-1β, IL-6, TNF-α andCOX2 mRNA, and surface expression of CD80, CD86 and MHC class IImolecules.

Example 7 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Human Bladder Smooth Muscle Cell ContractionExperimental Design

Cultured human bladder smooth muscle cells are exposed to inflammatorystimuli and non-inflammatory stimuli in the presence of analgesic agentand/or antimuscarinic agent at various concentrations. Thestimuli-induced muscle contraction is measured to evaluate theinhibitory effect of the analgesic agent and/or antimuscarinic agent.

The effectors, analgesic agents and antimuscarinic agents are describedin Example 2.

Human bladder smooth muscle cells are subjected to short term (1-2 hrs)or long term (24-48 hrs) stimulation of with:

(1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

Bladder smooth muscle cell contractions are recorded with a Grasspolygraph (Quincy Mass, USA).

Example 8 Effect of Analgesic Agents on Normal Human Bladder SmoothMuscle Cell Responses to Inflammatory and Non Inflammatory SignalsExperimental Design Culture of Normal Human Bladder Smooth Muscle Cells

Normal human bladder smooth muscle cells were isolated by enzymaticdigestion from macroscopically normal pieces of human bladder. Cellswere expended in vitro by culture at 37° C. in a 5% CO₂ atmosphere inRPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES, 2 mML-glutamine, 100 U/ml penicillin, and 100 mg/ml of streptomycin andpassage once a week by treatment with trypsin to detach cells followedby reseeding in a new culture flask. The first week of culture, theculture medium was supplemented with 0.5 ng/ml epidermal growth factor,2 ng/ml fibroblast growth factor, and 5 μg/ml insulin.

Treatment of Normal Human Bladder Smooth Muscle Cells with Analgesics InVitro

Bladder smooth muscle cells trypsinized and seeded in microcultureplates at a cell density of 3×10⁴ cells per well in 100 μl were treatedwith analgesic solutions (50 μl/well) either alone or together carbachol(10-Molar, 50 μl/well), as an example of non-inflammatory stimuli, orlipopolysaccharide (LPS) of Salmonella typhimurium (1 μg/ml, 50μl/well), as an example of non-inflammatory stimuli. When no othereffectors were added to the cells, 50 μl of RPMI 1640 without fetalbovine serum were added to the wells to adjust the final volume to 200μl.

After 24 hours of culture, 150 μl of culture supernatants werecollected, spun down for 2 min at 8,000 rpm at 4° C. to remove cells anddebris and stored at −70° C. for analysis of Prostaglandin E2 (PGE₂)responses by ELISA. Cells were fixed, permeabilized and blocked fordetection of COX2 using a fluorogenic substrate. In selected experimentscells were stimulated 12 hours in vitro for analysis of COX2, PGE2 andcytokine responses.

Analysis of COX2, PGE2 and Cytokine Responses

COX2 and PGE2 responses were analyzed as described in Example 3.Cytokine responses were analyzed as described in Example 2

Results

Analgesics Inhibit COX2 Responses of Normal Human Bladder Smooth MuscleCells to Inflammatory and Non-Inflammatory Stimuli—

Analysis of cells and culture supernatants after 24 hours of culturesshowed that none of the analgesics tested alone induced COX2 responsesin normal human bladder smooth muscle cells. However, as summarized inTable 6, carbachol induced low, but significant COX2 responses in normalhuman bladder smooth muscle cells. On the other hand, LPS treatmentresulted in higher levels of COX2 responses in normal human bladdersmooth muscle cells. Acetaminophen, aspirin, ibuprofen and naproxencould all suppress the effect of carbachol and LPS on COX2 levels. Thesuppressive effect of the analgesics was seen on LPS-induced responseswhen these drugs were tested at either 5 μM or 50 μM.

TABLE 6 COX2 expression by normal human bladder smooth muscle cellsafter in vitro stimulation with inflammatory and non-inflammatorystimuli and treatment with analgesic Total COX2 Total COX2 levels^(#)levels (Normalized (Normalized RFUs) RFUs) Stimuli Analgesic subject 1subject 2 None None 230 199 Carbachol 10⁻³M Acetaminophen (50 μM) 437462 Carbachol 10⁻³M Aspirin (50 μM) 298 310 Carbachol 10⁻³M Ibuprofen(50 μM) 312 297 Carbachol 10⁻³M Naproxen (50 μM) 309 330 Carbachol 10⁻³MAcetaminophen (50 μM) 296 354 LPS (10 μg/ml) None 672 633 LPS (10 μg/ml)Acetaminophen (5 μM) 428 457 LPS (10 μg/ml) Aspirin (5 μM) 472 491 LPS(10 μg/ml) Ibuprofen (5 μM) 417 456 LPS (10 μg/ml) Naproxen (5 μM 458501 LPS (10 μg/ml) Acetaminophen (50 μM) 399 509 LPS (10 μg/ml) Aspirin(50 μM) 413 484 LPS (10 μg/ml) Ibuprofen (50 μM) 427 466 LPS (10 μg/ml)Naproxen (50 μM) 409 458 ^(#)Data are expressed as mean of duplicates

Analgesics inhibit PGE2 responses of normal human bladder smooth musclecells to inflammatory and non-inflammatory stimuli—Consistent with theinduction of COX2 responses described above, both carbachol and LPSinduced production of PGE2 by normal human bladder smooth muscle cells.Acetaminophen, aspirin, ibuprofen and naproxen were also found tosuppress the LPS-induced PGE2 responses at either 5 μM or 50 μM (Table7).

TABLE 7 PGE2 secretion by normal human bladder smooth muscle cells afterin vitro stimulation with inflammatory and non-inflammatory stimuli andtreatment with analgesic PGE2 levels^(#) PGE2 levels (pg/ml) (pg/ml)Stimuli Analgesic Subject 1 Subject 2 None None <20.5 <20.5 Carbachol10⁻³M Acetaminophen (50 μM) 129 104 Carbachol 10⁻³M Aspirin (50 μM) 7662 Carbachol 10⁻³M Ibuprofen (50 μM) 89 59 Carbachol 10⁻³M Naproxen (50μM) 84 73 Carbachol 10⁻³M Acetaminophen (50 μM) 77 66 LPS (10 μg/ml)None 1125 998 LPS (10 μg/ml) Acetaminophen (5 μM) 817 542 LPS (10 μg/ml)Aspirin (5 μM) 838 598 LPS (10 μg/ml) Ibuprofen (5 μM) 824 527 LPS (10μg/ml) Naproxen (5 μM 859 506 LPS (10 μg/ml) Acetaminophen (50 μM) 803540 LPS (10 μg/ml) Aspirin (50 μM) 812 534 LPS (10 μg/ml) Ibuprofen (50μM) 821 501 LPS (10 μg/ml) Naproxen (50 μM) 819 523 ^(#)Data areexpressed as mean of duplicates

Analgesics Inhibit Cytokine Responses of Normal Human Bladder Cells toan Inflammatory Stimuli—

Analysis of cells and culture supernatants after 24 hours of culturesshowed that none the analgesics tested alone induced IL-6 or TNFαsecretion in normal human bladder smooth muscle cells. As shown inTables 8 and 9, the dose of carbachol tested induced low, butsignificant TNFα and IL-6 responses in normal human bladder smoothmuscle cells. On the other hand, LPS treatment resulted in massiveinduction of these proinflammatory cytokines. Acetaminophen, aspirin,ibuprofen and naproxen suppress the effect of carbachol and LPS on TNFαand IL-6 responses. The suppressive effect of the analgesic onLPS-induced responses was seen when these drugs were tested at either 5μM or 50 μM.

TABLE 8 TNFα secretion by normal human bladder smooth muscle cells afterin vitro stimulation with inflammatory and non-inflammatory stimuli andtreatment with analgesic TNFα TNFα (pg/ml)^(#) (pg/ml) Stimuli AnalgesicSubject 1 Subject 2 None None <5 <5 Carbachol 10⁻³M None 350 286Carbachol 10⁻³M Acetaminophen (50 μM) 138 164 Carbachol 10⁻³M Aspirin(50 μM) 110 142 Carbachol 10⁻³M Ibuprofen (50 μM) 146 121 Carbachol10⁻³M Naproxen (50 μM) 129 137 LPS (10 μg/ml) None 5725 4107 LPS (10μg/ml) Acetaminophen (5 μM) 2338 2267 LPS (10 μg/ml) Aspirin (5 μM) 24792187 LPS (10 μg/ml) Ibuprofen (5 μM) 2733 2288 LPS (10 μg/ml) Naproxen(5 μM 2591 2215 LPS (10 μg/ml) Acetaminophen (50 μM) 2184 2056 LPS (10μg/ml) Aspirin (50 μM) 2266 2089 LPS (10 μg/ml) Ibuprofen (50 μM) 26031997 LPS (10 μg/ml) Naproxen (50 μM) 2427 2192 ^(#)Data are expressed asmean of duplicates.

TABLE 9 IL-6 secretion by normal human bladder smooth muscle cells afterin vitro stimulation with inflammatory and non-inflammatory stimuli andtreatment with analgesic IL-6 IL-6 (pg/ml)^(#) (pg/ml) Stimuli AnalgesicSubject 1 Subject 2 None None <5 <5 Carbachol 10⁻³M None 232 278Carbachol 10⁻³M Acetaminophen (50 μM) 119 135 Carbachol 10⁻³M Aspirin(50 μM) 95 146 Carbachol 10⁻³M Ibuprofen (50 μM) 107 118 Carbachol 10⁻³MNaproxen (50 μM) 114 127 LPS (10 μg/ml) None 4838 4383 LPS (10 μg/ml)Acetaminophen (5 μM) 2012 2308 LPS (10 μg/ml) Aspirin (5 μM) 2199 2089LPS (10 μg/ml) Ibuprofen (5 μM) 2063 2173 LPS (10 μg/ml) Naproxen (5 μM2077 2229 LPS (10 μg/ml) Acetaminophen (50 μM) 2018 1983 LPS (10 μg/ml)Aspirin (50 μM) 1987 2010 LPS (10 μg/ml) Ibuprofen (50 μM) 2021 1991 LPS(10 μg/ml) Naproxen (50 μM) 2102 2028 ^(#)Data are expressed as mean ofduplicates

Primary normal human bladder smooth muscle cells were isolated, culturedand evaluated for their responses to analgesics in the presence ofnon-inflammatory (carbachol) and inflammatory (LPS) stimuli. The goal ofthis study was to determine whether or not normal human bladder smoothmuscle cells recapitulate the observations previously made with murinebladder cells.

The above-described experiment will be repeated with analgesic agentsand/or antimuscarinic agents in delayed-release, or extended-releaseformulation or delayed-and-extended-release formulations.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1-30. (canceled)
 31. A method for reducing the frequency of urination,comprising: administering to a subject who desires to reduce thefrequency of urination an effective amount of a pharmaceuticalcomposition comprising acetaminophen formulated in delayed-releaseformulation, wherein said acetaminophen is administered at a daily doseof 5 mg to 2000 mg.
 32. The method of claim 31, wherein saidacetaminophen is administered at a daily dose of 100 mg to 500 mg. 33.The method of claim 31, wherein said acetaminophen is administered at adaily dose of 500 mg to 2000 mg.
 34. The method of claim 31, whereinsaid acetaminophen is administered at a daily dose of 50 mg to 1600 mg.35. The method of claim 31, wherein said pharmaceutical compositionfurther comprises one or more antimuscarinic agents.
 36. The method ofclaim 31, wherein said pharmaceutical composition further comprises oneor more antidiuretics.
 37. The method of claim 31, wherein saidpharmaceutical composition further comprises one or more spasmolytics.38. The method of claim 31, wherein said delayed-release formulationcomprises a material selected from the group consisting of hydroxypropylmethylcellulose, methylcellulose, methyl hydroxyethylcellulose,hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers,polyethylene glycols, polyvinylpyrrolidone, methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and ethylcellulose.
 39. The method of claim 31,wherein said delayed-release formulation comprises glyceryl monostearateor glyceryl distearate.
 40. The method of claim 31, wherein saiddelayed-release formulation comprises a water insoluble capsule bodyclosed at one end with an insoluble, but permeable and swellablehydrogel plug, wherein said plug comprises a material selected from thegroup consisting of polymethacrylates, erodible compressed polymers,congealed melted polymer and enzymatically controlled erodible polymers.41. The method of claim 31, wherein said delayed-release formulationcomprises an enteric coating.
 42. The method of claim 31, wherein saidpharmaceutical composition further comprises one or more analgesicagents selected from the group consisting of aspirin, ibuprofen,naproxen, naproxen sodium, indomethacin and nabumetone.
 43. The methodof claim 31, wherein said pharmaceutical composition is administeredorally.
 44. The method of claim 31, wherein said pharmaceuticalcomposition is formulated for the oral administration.
 45. A method forreducing the frequency of urination, comprising: administering to asubject who desires to reduce the frequency of urination an effectiveamount of a pharmaceutical composition comprising acetaminophenformulated for immediate release, wherein said acetaminophen isadministered at a daily dose of 10 mg to 2000 mg.
 46. The method ofclaim 45, wherein said acetaminophen is administered at a daily dose of100 mg to 500 mg.
 47. The method of claim 45, wherein said acetaminophenis administered at a daily dose of 250 mg to 500 mg.
 48. The method ofclaim 45, wherein said acetaminophen is administered at a daily dose of250 mg to 1000 mg.
 49. The method of claim 45, wherein saidpharmaceutical composition further comprises one or more antimuscarinicagents selected from the group consisting of oxybutynin, solifenacin,darifenacin and atropine.
 50. The method of claim 45, wherein saidpharmaceutical composition further comprises one or more antidiuretics.51. The method of claim 45, wherein said pharmaceutical compositionfurther comprises one or more spasmolytics.
 52. The method of claim 45,wherein said pharmaceutical composition further comprises one or moreanalgesic agent selected from the group consisting of aspirin,ibuprofen, naproxen, naproxen sodium, indomethacin and nabumetone.
 53. Amethod for reducing the frequency of urination, comprising:administering to a subject who desires to reduce the frequency ofurination an effective amount of a pharmaceutical composition comprisingacetaminophen formulated in delayed-release formulation having animmediate-release component and a delayed-release component, whereinsaid acetaminophen is administered at a daily dose of 5 mg to 2000 mg.54. The method of claim 53, wherein said s delayed-release component mayalso be an extended release component that is delayed.
 55. The method ofclaim 53, wherein said pharmaceutical composition further comprises oneor more antimuscarinic agents in said immediate-release component, orsaid delayed-release component, or both components.
 56. The method ofclaim 53, wherein said pharmaceutical composition further comprises oneor more antidiuretics in said immediate-release component, or saiddelayed-release component, or both components.
 57. The method of claim53, wherein said pharmaceutical composition further comprises one ormore spasmolytics in said immediate-release component, or saiddelayed-release component, or both components.
 58. The method of claim53, wherein said pharmaceutical composition further comprises one ormore analgesic agents in said immediate-release component, or saiddelayed-release component, or both components, wherein said one or moreanalgesic agents are selected from the group consisting of aspirin,ibuprofen, naproxen, naproxen sodium, indomethacin and nabumetone. 59.The method of claim 53, wherein said pharmaceutical composition isadministered orally.
 60. The method of claim 53, wherein saidpharmaceutical composition is formulated for the oral administration.61. The method of claim 53, wherein said pharmaceutical compositionfurther comprises one or more analgesic agents in said immediate-releasecomponent, extended, or said delayed-release component, or anycombination of components.